Protease-Sensitive, VEGF-Mimetic Peptide, and IKVAV Laminin-Derived Peptide Sequences within Elastin-Like Recombinamer Scaffolds Provide Spatiotemporally Synchronized Guidance of Angiogenesis and Neurogenesis.

Spatiotemporal control of vascularization and innervation is a desired hallmark in advanced tissue regeneration. For this purpose, we design a 3D model scaffold, based on elastin-like recombinamer (ELR) hydrogels. This contains two interior and well-defined areas, small cylinders, with differentiated bioactivities with respect to the bulk. Both are constructed on a protease sensitive ELR with a fast-proteolyzed domain, but one bears a VEGF-mimetic peptide (QK) and the other a laminin-derived pentapeptide (IKVAV), to promote angiogenesis and neurogenesis, respectively. The outer bulk is based on a slow proteolytic sequence and RGD cell adhesion domains. In vitro studies show the effect of QK and IKVAV peptides on the promotion of endothelial cell and axon spreading, respectively. The subcutaneous implantation of the final 3D scaffold demonstrates the ability to spatiotemporally control angiogenesis and neurogenesis in vivo. Specifically, the inner small cylinder containing the QK peptide promotes fast endothelialization, whereas the one with IKVAV peptide promotes fast neurogenesis. Both, vascularization and innervation take place in advance of the bulk scaffold infiltration. This scaffold shows that it is possible to induce vascularization and innervation in predetermined areas of the scaffold well ahead to the bulk infiltration. That significantly increases the efficiency of the regenerative activity.

Fibrous Scaffolds From Elastin-Based Materials.

Current cutting-edge strategies in biomaterials science are focused on mimicking the design of natural systems which, over millions of years, have evolved to exhibit extraordinary properties. Based on this premise, one of the most challenging tasks is to imitate the natural extracellular matrix (ECM), due to its ubiquitous character and its crucial role in tissue integrity. The anisotropic fibrillar architecture of the ECM has been reported to have a significant influence on cell behaviour and function. A new paradigm that pivots around the idea of incorporating biomechanical and biomolecular cues into the design of biomaterials and systems for biomedical applications has emerged in recent years. Indeed, current trends in materials science address the development of innovative biomaterials that include the dynamics, biochemistry and structural features of the native ECM. In this context, one of the most actively studied biomaterials for tissue engineering and regenerative medicine applications are nanofiber-based scaffolds. Herein we provide a broad overview of the current status, challenges, manufacturing methods and applications of nanofibers based on elastin-based materials. Starting from an introduction to elastin as an inspiring fibrous protein, as well as to the natural and synthetic elastin-based biomaterials employed to meet the challenge of developing ECM-mimicking nanofibrous-based scaffolds, this review will follow with a description of the leading strategies currently employed in nanofibrous systems production, which in the case of elastin-based materials are mainly focused on supramolecular self-assembly mechanisms and the use of advanced manufacturing technologies. Thus, we will explore the tendency of elastin-based materials to form intrinsic fibers, and the self-assembly mechanisms involved. We will describe the function and self-assembly mechanisms of silk-like motifs, antimicrobial peptides and leucine zippers when incorporated into the backbone of the elastin-based biomaterial. Advanced polymer-processing technologies, such as electrospinning and additive manufacturing, as well as their specific features, will be presented and reviewed for the specific case of elastin-based nanofiber manufacture. Finally, we will present our perspectives and outlook on the current challenges facing the development of nanofibrous ECM-mimicking scaffolds based on elastin and elastin-like biomaterials, as well as future trends in nanofabrication and applications.

Elastin-Plasma Hybrid Hydrogels for Skin Tissue Engineering.

Dermo-epidermal equivalents based on plasma-derived fibrin hydrogels have been extensively studied for skin engineering. However, they showed rapid degradation and contraction over time and low mechanical properties which limit their reproducibility and lifespan. In order to achieve better mechanical properties, elasticity and biological properties, we incorporated a elastin-like recombinamer (ELR) network, based on two types of ELR, one modified with azide (SKS-N3) and other with cyclooctyne (SKS-Cyclo) chemical groups at molar ratio 1:1 at three different SKS (serine-lysine-serine sequence) concentrations (1, 3, and 5 wt.%), into plasma-derived fibrin hydrogels. Our results showed a decrease in gelation time and contraction, both in the absence and presence of the encapsulated human primary fibroblasts (hFBs), higher mechanical properties and increase in elasticity when SKSs content is equal or higher than 3%. However, hFBs proliferation showed an improvement when the lowest SKS content (1 wt.%) was used but started decreasing when increasing SKS concentration at day 14 with respect to the plasma control. Proliferation of human primary keratinocytes (hKCs) seeded on top of the hybrid-plasma hydrogels containing 1 and 3% of SKS showed no differences to plasma control and an increase in hKCs proliferation was observed for hybrid-plasma hydrogels containing 5 wt.% of SKS. These promising results showed the need to achieve a balance between the reduced contraction, the better mechanical properties and biological properties and indicate the potential of using this type of hydrogel as a testing platform for pharmaceutical products and cosmetics, and future work will elucidate their potential.

Controlled Production of Elastin-like Recombinamer Polymer-Based Membranes at a Liquid-Liquid Interface by Click Chemistry.

Diffusion of organic and inorganic molecules controls most industrial and biological processes that occur in a liquid phase. Although significant efforts have been devoted to the design and operation of large-scale purification systems, diffusion devices with adjustable biochemical characteristics have remained difficult to achieve. In this regard, micrometer-scale, bioinspired membranes with tunable diffusion properties have been engineered by covalent cross-linking of two elastin-like recombinamers (ELRs) at a liquid-liquid interface. The covalent approach selected provides the desired ELR-based membranes with structural support, and modulation of the concentration of the polypeptides employed confers direct control of the thickness, pore size, and diffusive properties over a broad range of molecular weights (4-150 kDa). The recombinant and versatile nature of the proteinaceous building blocks employed further paves the way to engineering bioactive motifs within the membrane scaffold, thereby widening their applicability in the biological field.

Elastin-Based Materials: Promising Candidates for Cardiac Tissue Regeneration.

Stroke and cardiovascular episodes are still some of the most common diseases worldwide, causing millions of deaths and costing billions of Euros to healthcare systems. The use of new biomaterials with enhanced biological and physical properties has opened the door to new approaches in cardiovascular applications. Elastin-based materials are biomaterials with some of the most promising properties. Indeed, these biomaterials have started to yield good results in cardiovascular and angiogenesis applications. In this review, we explore the latest trends in elastin-derived materials for cardiac regeneration and the different possibilities that are being explored by researchers to regenerate an infarcted muscle and restore its normal function. Elastin-based materials can be processed in different manners to create injectable systems or hydrogel scaffolds that can be applied by simple injection or as patches to cover the damaged area and regenerate it. Such materials have been applied to directly regenerate the damaged cardiac muscle and to create complex structures, such as heart valves or new bio-stents that could help to restore the normal function of the heart or to minimize damage after a stroke. We will discuss the possibilities that elastin-based materials offer in cardiac tissue engineering, either alone or in combination with other biomaterials, in order to illustrate the wide range of options that are being explored. Moreover, although tremendous advances have been achieved with such elastin-based materials, there is still room for new approaches that could trigger advances in cardiac tissue regeneration.

An elastin-like recombinamer-based bioactive hydrogel embedded with mesenchymal stromal cells as an injectable scaffold for osteochondral repair.

The aim of this study was to evaluate injectable, in situ cross-linkable elastin-like recombinamers (ELRs) for osteochondral repair. Both the ELR-based hydrogel alone and the ELR-based hydrogel embedded with rabbit mesenchymal stromal cells (rMSCs) were tested for the regeneration of critical subchondral defects in 10 New Zealand rabbits. Thus, cylindrical osteochondral defects were filled with an aqueous solution of ELRs and the animals sacrificed at 4 months for histological and gross evaluation of features of biomaterial performance, including integration, cellular infiltration, surrounding matrix quality and the new matrix in the defects. Although both approaches helped cartilage regeneration, the results suggest that the specific composition of the rMSC-containing hydrogel permitted adequate bone regeneration, whereas the ELR-based hydrogel alone led to an excellent regeneration of hyaline cartilage. In conclusion, the ELR cross-linker solution can be easily delivered and forms a stable well-integrated hydrogel that supports infiltration and de novo matrix synthesis.

Silk-ELR co-recombinamer covered stents obtained by electrospinning.

In the field of tissue engineering the choice of materials is of great importance given the possibility to use biocompatible polymers produced by means of biotechnology. A large number of synthetic and natural materials have been used to this purpose and processed into scaffolds using Electrospinning technique. Among materials that could be used for the fabrication of scaffold and degradable membranes, natural polymers such as collagen, elastin or fibroin offer the possibility to design structures strictly similar to the extracellular matrix (ECM). Biotechnology and genetic engineering made possible the advent of a new class of biopolymers called protein-based polymers. One example is represented by the silk-elastin-proteins that combine the elasticity and resilience of elastin with the high tensile strength of silk-fibroin and display engineered bioactive sequences. In this work, we use electrospinning technique to produce a fibrous scaffold made of the co-recombinamer Silk-ELR. Obtained fibres have been characterized from the morphological point of view. Homogeneity and morphology have been explored using Scanning Electron Microscopy. A thorough study regarding the influence of Voltage, flow rate and distance have been carried out to determine the appropriate parameters to obtain the fibrous mats without defects and with a good distribution of diameters. Cytocompatibility has also been in vitro tested. For the first time we use the co-recombinamer Silk-ELR for the fabrication of a 2.5 angioplasty balloon coating. This structure could be useful as a coated scaffold for the regeneration of intima layer of vessels.

Random and oriented electrospun fibers based on a multicomponent, in situ clickable elastin-like recombinamer system for dermal tissue engineering.

Herein we present a system to obtain fibers from clickable elastin-like recombinamers (ELRs) that crosslink in situ during the electrospinning process itself, with no need for any further treatment to stabilize them. These ELR-click fibers are completely stable under in vitro conditions. A wrinkled fiber morphology is obtained. In addition to a random fiber orientation, oriented fibers with a high degree of alignment and coherence can also be obtained by using a rotational electrode. The production of multicomponent fibers means that different functionalities, such as cell-adhesion domains (RGD peptides), can be incorporated into them. In a subsequent study, two main cell lines present in the dermis and epidermis, namely keratinocytes and fibroblasts, were cultured on top of the ELR-click fibers. Adhesion, proliferation, fluorescence, immunostaining and histology studies showed the cytocompatibility of these scaffolds, thus suggesting their possible use for wound dressings in skin tissue engineering applications. STATEMENT OF SIGNIFICANCE: For the first time stable electrospun bioactive fibers are obtained by the in situ mixing of two "clickable" ELR components previously described by Gonzalez et al (Acta Biomaterialia 2014). This work describes an efficient system to prepare fibrous scaffolds based on peptidic polymers by electrospinning without the need of crosslinking agents that could be harmful for cells or living tissues. These bioactive fibers support cell growth due to the inclusion of RGD motifs (Staubli et al. Biomaterials 2017). Finally, the in vitro biocompatibility of the two main cell types found in the outer layers of skin, fibroblasts and keratinocytes, indicates that this system is of great interest to prepare elastic artificial skin substitutes for wound healing applications.

Biocompatibility of two model elastin-like recombinamer-based hydrogels formed through physical or chemical cross-linking for various applications in tissue engineering and regenerative medicine.

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein-based ones, such as elastin-like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix-like hydrogels through either physical or chemical cross-linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase-expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR-based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL-1ß, IL-4, IL-6, and IL-10 concentrations were measured by enzyme-linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine-related applications.

Control of angiogenesis and host response by modulating the cell adhesion properties of an Elastin-Like Recombinamer-based hydrogel.

The control of the in vivo vascularization of engineered tissue substitutes is essential in order to obtain either a rapid induction or a complete inhibition of the process (e.g. in muscles and hyaline-cartilage, respectively). Among the several polymers available, Elastin-Like Recombinamers (ELR)-based hydrogel stands out as a promising material for tissue engineering thanks to its viscoelastic properties, non-toxicity, and non-immunogenicity. In this study, we hypothesized that varying the cell adhesion properties of ELR-hydrogels could modulate the high angiogenic potential of adipose tissue-derived stromal vascular fraction (SVF) cells, predominantly composed of endothelial/mural and mesenchymal cells. Human SVF cells, embedded in RGD-REDV-bioactivated or unmodified ELR-hydrogels, were implanted in rat subcutaneous pockets either immediately or upon 5-day-culture in perfusion-bioreactors. Perfusion-based culture enhanced the endothelial cell cord-like-organization and the release of pro-angiogenic factors in functionalized constructs. While in vivo vascularization and host cell infiltration within the bioactivated gels were highly enhanced, the two processes were strongly inhibited in non-functionalized SVF-based hydrogels up to 28 days. ELR-based hydrogels showed a great potential to determine the successful integration of engineered substitutes thanks to their capacity to finely control the angiogenic/inflammation process at the recipient site, even in presence of SVF cells.

Hybrid elastin-like recombinamer-fibrin gels: physical characterization and in vitro evaluation for cardiovascular tissue engineering applications.

In the field of tissue engineering, the properties of the scaffolds are of crucial importance for the success of the application. Hybrid materials combine the properties of the different components that constitute them. In this study hybrid gels of Elastin-Like Recombinamer (ELR) and fibrin were prepared with a range of polymer concentrations and ELR-to-fibrin ratios. The correlation between SEM micrographs, porosities, swelling ratios and rheological properties was discussed and a poroelastic mechanism was suggested to explain the mechanical behavior of the hybrid gels. Applicability as scaffold materials for cardiovascular tissue engineering was shown by the realization of cell-laden matrixes which supported the synthesis of collagens as revealed by immunohistochemical analysis. As a proof of concept, a tissue-engineered heart valve was fabricated by injection moulding and cultivated in a bioreactor for 3 weeks under dynamic conditions. Tissue analysis revealed the production of collagen I and III, fundamental proteins for cardiovascular constructs.

Multiple-Step Injection Molding for Fibrin-Based Tissue-Engineered Heart Valves.

Heart valves are elaborate and highly heterogeneous structures of the circulatory system. Despite the well accepted relationship between the structural and mechanical anisotropy and the optimal function of the valves, most approaches to create tissue-engineered heart valves (TEHVs) do not try to mimic this complexity and rely on one homogenous combination of cells and materials for the whole construct. The aim of this study was to establish an easy and versatile method to introduce spatial diversity into a heart valve fibrin scaffold. We developed a multiple-step injection molding process that enables the fabrication of TEHVs with heterogeneous composition (cell/scaffold material) of wall and leaflets without the need of gluing or suturing components together, with the leaflets firmly connected to the wall. The integrity of the valves and their functionality was proved by either opening/closing cycles in a bioreactor (proof of principle without cells) or with continuous stimulation over 2 weeks. We demonstrated the potential of the method by the two-step molding of the wall and the leaflets containing different cell lines. Immunohistology after stimulation confirmed tissue formation and demonstrated the localization of the different cell types. Furthermore, we showed the proof of principle fabrication of valves using different materials for wall (fibrin) and leaflets (hybrid gel of fibrin/elastin-like recombinamer) and with layered leaflets. The method is easy to implement, does not require special facilities, and can be reproduced in any tissue-engineering lab. While it has been demonstrated here with fibrin, it can easily be extended to other hydrogels.

Nanogel formation from dilute solutions of clickable elastin-like recombinamers and its dependence on temperature: two fractal gelation modes.

Diluted, complementary, click-reactive elastin-like recombinamer (ELR) solutions have been prepared and mixed at two different temperatures, one below and one above the characteristic transition temperature (Tt) of these chemically modified ELRs. FTIR measurements, size, aspect ratio, zeta potential, and microrheological measurements have been carried out on the nanostructures formed under these dilute conditions as a way to better understand the relationship between the final macroscopic properties of ELR-based hydrogels and the molecular conditions governing the initial stages of the chemical cross-linking process that occurs, especially its dependence on the preparation temperature relative to Tt. As a result, two different fractal modes of gel formation have been found at the two temperatures studied (above and below Tt). Thus, when the reaction mixture is prepared below Tt, essentially one-dimensional linear nanogels with a high aspect ratio are obtained. In contrast, 3D nanogels are formed above Tt, with spherical shapes predominating. These different structures seem to reflect the two molecular organizations of the single components of the mixture under these conditions, namely extended chains below Tt and a spherical arrangement above Tt. In addition to the interest in these nanogels as models for understanding the formation of microscopic structures and differential macroscopic properties under more conventional hydrogel-formation conditions, these nanogels are of interest because of their thermoresponsiveness and biocompatibility, which provide them with potential uses for drug delivery and other biomedical applications in living systems.

Elastin-like recombinamer catalyst-free click gels: characterization of poroelastic and intrinsic viscoelastic properties.

Elastin-like recombinamer catalyst-free click gels (ELR-CFCGs) have been prepared and characterized by modifying both a structural ELR (VKVx24) and a biofunctionalized ELR-bearing RGD cell-adhesion sequence (HRGD6) to bear the reactive groups needed to form hydrogels via a click reaction. Prior to formation of the ELR-CFCGs, azide-bearing and cyclooctyne-modified ELRs were also synthesized. Subsequent covalent crosslinking was based on the reaction between these azide and cyclooctyne groups, which takes place under physiological conditions and without the need for a catalyst. The correlation among SEM micrographs, porosity, swelling ratio, and rheological measurements have been carried out. The storage and loss moduli at 1Hz are in the range 1-10kPa and 100-1000Pa, respectively. The linear dependence of |G∗| on f(½) and the peak value of tan δ were considered to be consistent with a poroelastic mechanism dominating the frequency range 0.3-70Hz. The discrete relaxation spectrum was obtained from stress relaxation measurements (t>5s). The good fit of the relaxation modulus to decrease exponential functions suggests that an intrinsic viscoelastic mechanism dominates the transients. Several recombinamer concentrations and temperatures were tested to obtain gels with fully tuneable properties that could find applications in the biomedical field.